Abstract Dopaminergic transmission in the synapse is primarily terminated by removal of dopamine (DA) into presynaptic nerve terminals via the dopamine transporter (DAT), one of a superfamily of Na+/Cl--dependent neurotransmitter transporters. DAT is a major target of psychostimulants such as amphetamine (AMPH) and cocaine. Dysfunctional DAT has been implicated in neurodegenerative and neuropsychiatric disorders such as Parkinson disease, schizophrenia, attention deficit and hyperactivity disorder, and drug addiction. We have demonstrated a novel role of protein kinase C[unreadable] (PKC[unreadable]) in regulation of basal and substrate-induced DAT trafficking/activity. Interestingly, PKC[unreadable] regulation of DAT trafficking/activity seems to involve two other DAT directly interacting proteins RACK1 and D2 dopamine receptor (D2R). RACK1 enhances AMPH-induced DA efflux when co-expressed with PKC[unreadable]II in a human DAT (hDAT)-stably expressing mouse mesencephalic dopaminergic cell line (MN9D). Activation of D2R increases DAT trafficking/activity in PKC[unreadable] WT (PKC[unreadable]+/+) mice but not in PKC[unreadable] knockout (PKC[unreadable]-/-)mice. These data suggest that RACK1 and D2R may be important intermediate players for facilitation of PKC[unreadable] effect on DAT regulation. The overall objective of the proposal is to characterize roles of RACK1 and D2R in PKC[unreadable] regulation of basal and induced DAT trafficking and activity. The specific aims of this proposal are to test the hypothesis that 1) RACK1 facilitates PKC[unreadable] coupling to DAT and its regulatory effect on basal and substrate-induced DAT trafficking and activity;(2) D2R-induced DAT trafficking requires PKC[unreadable] and involves trafficking of the D2R-DAT complex upon D2R activation. These hypotheses will be tested using overexpression, knockdown, or knockout of targeted proteins in MN9D cells and/or PKC[unreadable]+/+ and PKC[unreadable]-/- mice. This project will provide a new molecular and cellular avenue for potential drug intervention to drug addiction, psychiatric, and neurodegenerative diseases by targeting RACK1, D2R, and PKC[unreadable] as modulators of dopaminergic homeostasis.